This project concerns the molecular and cellular pathophysiology of sickle cell disease and related red cell disorders, with a major focus on the origin of dense, dehydrated SS cells in the circulation, and emphasis on basic issues relevant to other red cell disorders and cell physiology: I. How does sickling act on SS reticulocyte (retic) heterogeneity to generate different mature red cell subpopulations? II. What is the nature of the sickling-induced permeability pathway and how does it produce the observed abnormalities of ion transport and content? III. How does the formation, breakdown and possible structural variations of deoxy-Hb S polymers in SS cells directly alter cell volume, ion distribution, and metabolism? Studies to these ends will: I. Develop further our hypothesis of a direct retic origin of most dense SS cells: use simulations of our new non-steady-state red cell and retic models to predict conditions to separate SS retics with different transport properties; identify their transport heterogeneities, Ca2= and Mg2= metabolism, and our newly found Ca2=-sensitive Cl permeability; and assess the role of "stress retics" in the dehydration process, and in dense cell variations in vasoocclusive sickle crises; II. Study the sickling-induced permeability pathway ("Psickle") and the mechanisms of ion, pH and volume abnormalities in SS retics and older cells; characterize our newly found heparin effect of a magnified Psickle-Na/K in the absence of Ca2+, for transport and ultrastructural studies of the leak; fluorescence-image single cells containing Ca2+ chelators, to locate the Ca2+ leaks and the distribution of Pca and steady state [Ca2+] in normal and sickle cells; measure what pO2's and polymer fractions are needed to permeabilize different density S cells, testing the hypothesis that dense SS cells may be permeabilized most of the time in the circulation; test for K:C1 cotransport in inside-out vesicles from retics and mature red cells, and if it can be activated by exposure to Hb S or C in vitro; test whether our newly found increased inosine monophosphate in SS cells reflects their exposure to [Ca2+]; and study metHb and hemichrome formation in SS fractions using electron paramagnetic resonance. III. Apply new accurate methods to (i) estimate the concentration of Hb in the polymer, Cp; (ii) test whether Cp changes with cell factors affecting polymer solubility (C); (iii) estimate the incorporation of Hbs a, F and C into the polymer (as hybrids, tetramers, and in T or R conformations), and incorporation of low and intermediate MW substances into the polymer-associates water compartment (PWC, derived from Cp) which excludes soluble macromolecules. We can then: predict the osmotic effects of polymerization as a function of cell MCHC, and test the predictions directly; with Cp and non-S Hb incorporation measured, study polymer ultrastructure by electronmicroscopy, and assess glycolytic effects of enzyme-substrate redistribution due to polymerization in dense SS cells.